| Aim:To investigate the effects of fucoidan polysaccharide sulfate(FPS)on renal injury in rats with uric acid nephropathy(UAN).Method:1.In vivo study on the protective effect of FPS on renal tubular epithelial cells in UAN rats1.1 UAN rat model constructionThe model group received intragastric administration of potassium oxonate(250 mg/kg)combined with adenine(100 mg/kg)while the normal control group was given equal doses of distilled water once daily for 14 days.2.1 The effects of FPS on renal tubular injury in UAN ratsThe effect of FPS on potassium oxonate combined with adenine-induced UAN rats:60 SD rats were randomly divided into normal control group,model group,fucoidan low-dose,medium dose,high-dose group and sacrificed on the 14th day.Serum uric acid,urea nitrogen,and creatinine levels were observed in all groups.Pathological changes of renal tubules were observed by HE staining.Microstructure changes of renal tubular epithelial cells were detected by transmission electron microscope.Expression of ROS in kidneys was detected by ELISA.Apoptosis of renal tubular epithelial cells was measured by TUNEL,and the expression of LC3B in kidney was detected by immunofluorescence.2.In vivo study on the effects of injury reduced by FPS in high uric acid-induced tubular epithelial cell2.1 Establishment of tubular epithelial cell injury model with high uric acid2.1.1The effects of different concentrations of UA on the proliferation of renal tubular epithelial cells(NRK-52E)at different time points:In vitro NRK-52E cells were seeded on 96-well plates at 4000 cells per well and divided into five groups:Standard group,180umol/L,90umol/L,45umol/L,22.5umol/L.Blank wells were set up.5 wells were set in each group.CCK8 method was used to detect cell viability after 12h,24h and 48h.2.1.2 The effects of different concentrations of FPS on the proliferation of NRK-52E cells induced by specific UA concentrations at different time points:NRK-52E cells were seeded on 96-well plates at 4000 cells per well.Due to different concentrations of FPS intervention,NRK-52E cells divided into five groups:UA(180umol/L),200ug/ml,100ug/ml,50ug/ml,25ug/ml.Each group was stimulated with UA(180umol/L),and blank wells were set up.After cultured for 12h,24h and 48h,cell proliferation was detected by CCK8.2.2 The effects of FPS on apoptosis of NRK-52E cells induced by HUANRK-52E cells were randomly divided into four groups:normal group,uric acid group,FPS low-dose group,FPS middle-dose group,and FPS high-dose group.The expression of ROS was detected by ELISA,and apoptosis was detected by flow cytometry after cultivate for 24h.2.3 The effects of FPS on autophagy of NRK-52E cells induced by HUA NRK-52E cells were randomly divided into four groups:normal group,uric acid group,FPS low-dose group,FPS middle-dose group,and FPS high-dose group.After cultivate for 24h,transmission electron microscopy was used to observe the changes of autophagosomes and mitochondria,immunofluorescence was used to detect the expression of LC3B,and the expression of Parkin,Pink and LC3BII proteins were measured by western blot.Results:1.In vivo study on the protective effect of FPS on renal tubular epithelial cells in UAN rats1.1 UAN rat model constructionResults showed that serum UA,BUN,and Cr in the model group were significantly higher than those in the normal control group,indicating successful model establishment.2.1 The effects of FPS on renal tubular injury in UAN ratsResults showed that compared with the model group,serum UA,BUN and Cr were significantly lower in the FPS treatment group and allopurinol group.HE staining showed that some renal tubules in the kidney of the model group were completely destroyed,and a large number of yellow-brown urate crystals were deposited.The renal tubules in the FPS and allopurinol groups were significantly improved.Transmission electron microscopy showed that the microvilli of the renal tubular epithelial cells in the model group were disordered and dissolving,and a large number of mitochondria,lysosomes,and autophagosomes were observed in the basement.The level of ROS in the kidneys of the model group increased,and the expression of ROS in the kidneys of the FPS group decreased significantly.The TUNEL assay showed that compared with the normal control group,the apoptosis of renal tubular epithelial cells in the model group was significantly increased,and the apoptotic cells in the FPS treatment group and allopurinol group were significantly reduced.Immunofluorescence assay showed that LC3B was highly expressed in the renal cells of the model group,and the expression of LC3B in the renal cells of the FPS-treated group was significantly decreased.2.In vivo study on the effects of injury reduced by FPS in high uric acid-induced tubular epithelial cell2.1 Establishment of tubular epithelial cell injury model with high uric acid2.1.1 CCK8 results showed that compared with the standard group,when the uric acid concentration was 180umol/L,and the stimulation time was 24h,the proliferation of NRK-52E cells obviously decreased.2.1.2 CCK8 results showed that the proliferation of NRK-52E cells were significantly improved compared with the UA group,when FPS concentrations were 200,100,and 50 ?g/ml,respectively,and the intervention time was 24 h.2.2 The effects of FPS on apoptosis of NRK-52E cells induced by HUACompared with the normal group,the ROS content in the uric acid group was significantly increased,while the FPS treatment group was significantly decreased compared with the uric acid group,and there was a dose-dependent relationship.Flow cytometry showed that apoptosis was significantly greater in the uric acid group than in the normal group,and FPS treatment significantly improved apoptosis of NRK-52E cells.2.3 The effects of FPS on autophagy of NRK-52E cells induced by HUATransmission electron microscopy revealed that the uric acid group had severely damaged cell membranes,swelling of mitochondria,increased autophagosomes,decreased membrane structure and mitochondrial damage in the FPS group,and decreased autophagosomes.Immunofluorescence showed that compared with the normal group,the expression of LC3B in the uric acid group increased while the one in FPS treatment group was significantly decreased.Western Blot showed that the expression of Parkin,Pink and LC3BII protein were significantly increased in uric acid group while FPS treatment group can significantly reduce the high expression of those induced by uric acid.Conclusion:1.Potassium oxonate combined with adenine to induce uric acid nephropathy rat model2.The occurrence and development of UAN is associated with increased expression of ROS in renal tubular epithelial cells,mitochondrial damage,and excessive autophagy.FPS can improve the damage of UAN renal tubular epithelial cells. |
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